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The Journal of Immunology, 1999, 162: 6016-6023.
Copyright © 1999 by The American Association of Immunologists

Signaling Through the Lymphotoxin-ß Receptor Stimulates HIV-1 Replication Alone and in Cooperation with Soluble or Membrane-Bound TNF-{alpha}1

William L. Marshall{dagger}, Brigitta M. N. Brinkman*, Christine M. Ambrose{ddagger}, Patricia A. Pesavento*, Adele M. Uglialoro*, Edna Teng{dagger}, Robert W. Finberg{dagger}, Jeffrey L. Browning{ddagger} and Anne E. Goldfeld2,{dagger}

* Center for Blood Research and {dagger} Division of Adult Oncology, Dana-Farber Cancer Institute, Boston, MA 02115; and {ddagger} Biogen, Inc., Cambridge, MA 02138


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The level of ongoing HIV-1 replication within an individual is critical to HIV-1 pathogenesis. Among host immune factors, the cytokine TNF-{alpha} has previously been shown to increase HIV-1 replication in various monocyte and T cell model systems. Here, we demonstrate that signaling through the TNF receptor family member, the lymphotoxin-ß (LT-ß) receptor (LT-ßR), also regulates HIV-1 replication. Furthermore, HIV-1 replication is cooperatively stimulated when the distinct LT-ßR and TNF receptor systems are simultaneously engaged by their specific ligands. Moreover, in a physiological coculture cellular assay system, we show that membrane-bound TNF-{alpha} and LT-{alpha}1ß2 act virtually identically to their soluble forms in the regulation of HIV-1 replication. Thus, cosignaling via the LT-ß and TNF-{alpha} receptors is probably involved in the modulation of HIV-1 replication and the subsequent determination of HIV-1 viral burden in monocytes. Intriguingly, surface expression of LT-{alpha}1ß2 is up-regulated on a T cell line acutely infected with HIV-1, suggesting a positive feedback loop between HIV-1 infection, LT-{alpha}1ß2 expression, and HIV-1 replication. Given the critical role that LT-{alpha}1ß2 plays in lymphoid architecture, we speculate that LT-{alpha}1ß2 may be involved in HIV-associated abnormalities of the lymphoid organs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The HIV-1 viral burden within an infected individual is a key factor both in the progression of HIV-1 infection to clinical AIDS (1, 2), and in the perinatal transmission of HIV-1 from mother to fetus (3, 4, 5). The viral set-point within an individual is a function of both viral elimination by the host immune system and the ongoing level of HIV-1 replication (6). Although host immune factors are involved in both these processes, to date they remain poorly defined, particularly with regard to those factors that regulate HIV-1 replication and thus viral load (reviewed in 7).

Among host immune factors, the cytokine TNF-{alpha} has been shown to increase HIV-1 replication in various monocyte and T cell model systems (8, 9). TNF-{alpha} exists as both a membrane-bound 26-kDa form and a 17-kDa soluble form (10) and signals through two receptors, p55 and p75 (reviewed in 11). The two TNF receptors induce both specific and overlapping responses, including activation of the transcription factor, NF-{kappa}B (12, 13, 14, 15). It has been suggested that the soluble and membrane forms of TNF-{alpha} signal preferentially through p55 and p75 (16).

The membrane-anchored lymphotoxin-{alpha}1ß2 (LT-{alpha}1ß2)3 ligand molecule, which is expressed on the surface of activated T and B cells (17, 18), but not on the surface of cells of nonlymphoid origin, binds to the LT-ßR. The LT-ßR is expressed on the surface of some monocytic cells (18, 19), some dendritic cells (20), and the promyelocyctic U937 cell line (18) (our unpublished observations), but not on T or B cells. The LT-ßR signals through a pathway that is distinct from the TNF receptor signaling pathways (reviewed in 21) and is involved in the development of lymph nodes, the maintenance of lymphoid architecture, and the function of follicular dendritic cells (18, 21, 22). An additional ligand of unknown biological function called LIGHT can also bind the LT-ßR (23).

Multiple studies have also underscored the importance of TNF-{alpha} in lymphoid biology. Mice deficient in TNF-{alpha} lack germinal center formation in splenic follicles (24, 25, 26). Furthermore, TNF-{alpha} is involved in the generation of follicular dendritic cells (27) and B cell proliferation (28). Thus, given that disruption of lymphoid architecture is a hallmark of HIV-1 infection (29, 30), LT-{alpha}1ß2 and TNF-{alpha} are particularly interesting candidate host factors to study in relation to HIV-1 disease progression and pathogenesis.

The monocyte plays a critical role in the progression of HIV-1 infection. HIV-1-infected monocytes are numerous early in HIV-1 infection and are a major source of virus in advanced AIDS when the T cell population has been depleted (30, 31, 32). The host factors involved in the regulation of HIV-1 replication in monocytes are therefore of particular importance in the establishment of HIV-1 infection, viral burden, and disease progression.

To explore the role of TNF-{alpha} and LT-{alpha}1ß2 in the regulation of HIV-1 in infected monocytes, we used the chronically HIV-1-infected U1 cell line, which is the best characterized reproducible in vitro monocytic system used to study cytokine-mediated HIV-1 replication (9, 33, 34). Here, we show that costimulation through LT-ßR and TNF receptors results in a dramatic increase in viral replication in U1 cells. Furthermore, cocultivation of cells expressing membrane-bound forms of LT-{alpha}1ß2 and TNF-{alpha} with U1 cells or the clustering of the LT-ßR and TNFp75 receptors on U1 cells also greatly increases viral replication. These studies thus indicate that signaling through the LT-ß and TNF receptors in monocytes may influence HIV-1 viral burden and disease progression. Furthermore, they suggest that strategies that inhibit TNF-{alpha} and LT-{alpha}1ß2 expression and signaling may be useful in decreasing HIV-1 viral burden and thus disease progression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

The following reagents were obtained from the AIDS Reference Reagents Program: CEM, Jurkat, and H9 cell lines. The II-23 cell line was previously described (14). The U1 cell line (34) undergoes a time-dependent decay in the ability of TNF-{alpha} to induce HIV-1 replication as noted previously (33, 34). We obtained U1 cells from the AIDS Repository, and U1 clones were isolated by limiting dilution and then selected for maximal responsiveness to TNF-{alpha}. More than one clone was used in the experiments described with the U1 cell line, and results were comparable between clones. Cells were maintained in RPMI 1640 supplemented with 10% FBS with L-glutamine and penicillin/streptomycin (Life Technologies, Gaithersburg, MD).

H9 cells were infected with the HIV-1MN strain at a multiplicity of infection of 1.0 as previously described (35, 36) and were analyzed by flow cytometry for expression of surface LT-{alpha}1ß2 or TNF-{alpha}. Cells were preincubated with human serum for 20 min at 4°C, and all subsequent incubations were performed at 4°C for 40 min each. Cells were incubated with LT-ßR-Fc or with the anti-TNF-{alpha} Ab cA2 (see Reagents below). As a negative control, some cells were incubated with human IgG1. This was followed by incubation with FITC-labeled goat anti-human (F(ab')2 (BioSource, Camarillo, CA). The Abs NC2 and CH12 (see Reagents below) were used as previously described (37).

The human embryonic kidney cell line E293, stably transfected with EBV nuclear Ag, was obtained from Invitrogen (Carlsbad, CA) and maintained in DMEM supplemented with 10% FBS, L-glutamine, penicillin/streptomycin (Life Technologies), and 250 µg/ml G418.

The p24 concentration was determined by ELISA according to the manufacturer’s specifications (New England Nuclear-DuPont, Boston, MA).

Reagents

The properties of the recombinant LT-{alpha}1ß2 ligand have been described previously (38). The anti-LT-ßR Ab, CBE-11 (39); the anti-TNF-R75 Ab (a gift from W. Lesslauer, Hoffmann-La Roche, Basel, Switzerland) (40); and the anti-very late Ag-2 Ab, DE9 (a gift from J. Bergelson) (41) have all been previously described. The Abs NC2 and CH12, which respectively recognized either all LT-{alpha}-containing forms or only LT-{alpha}2ß1 forms of surface LT, have also been described previously (37). The control Abs MOPC 21 (a murine IgG1) and human IgG1 were obtained from Sigma (St. Louis, MO). Recombinant TNF-{alpha} was purchased from Genzyme (Cambridge, MA). The anti-TNF-{alpha} Ab, cA2, and the isotype-matched control Ab were gifts from Centocor (Malverne, CA).

The LT-ßR-Fc and TNF-R55-Fc fusion proteins have been described previously (37). Receptor-Fc fusion proteins can lead to FcR binding, which can, in turn, stimulate monocytes to produce HIV-1 (42), thereby complicating the analysis of inhibition by the fusion proteins. To ensure that Fc receptor-mediated events did not complicate the analysis of specificity, in some experiments an LT-ßR-Fc construct lacking the CH2 domain was employed as a control. This construct cannot bind Fc receptors or fix complement, and its origin has been previously described (37). CHO cells were transfected with the resulting construct, and purified CH2-less LT-ßR-Fc was purified from the CHO cell supernatants as previously described (37).

Receptor cross-linking experiments

Affinity-purified goat anti-IgG Fc (Jackson ImmunoResearch Laboratories, West Grove, PA) was immobilized on 24-well plates as described previously (39, 43) and was incubated with the Abs noted in the figures. U1 cells were plated on the washed plates at 2 x 105 cells/ml, and p24 values were measured from supernatants at 48 h.

EMSA

U1 cells were incubated for 2 h with or without 100 U/ml TNF-{alpha} or LT-{alpha}1ß2 at 50 ng/ml where indicated, nuclear extracts were prepared, and EMSAs were performed as previously described (44). The synthetic oligonucleotides used in the EMSA were: HIV-1-NF-{kappa}B, 5'-TCGACCGAGTGGGGACTTTCCTCTG-3' (-61 to -27 nucleotides) and 5'-GTTGAATGATTCTTTCCCCGCCCTCCTCTCGCCCCAGGGACA-3'.

DNA constructs and transfection

The derivation of the vectors used for the expression of LT-{alpha}D50N and LT-ß in E293 cells has been described (45). The wild-type human TNF-{alpha} gene was amplified from an activated cDNA library derived from II-23 cells using PCR primers designed to complement the 12 nucleotides or the last 12 nucleotides of the cDNA encoding TNF-{alpha}. The mutant membrane-retained form of human TNF-{alpha} was constructed from a genomic clone containing deletion of the first 12 TNF-{alpha} amino acids (TNF({Delta}1–12); a gift from G. Kollias) from which the TNF-{alpha} promoter was excised. The wild type TNF-{alpha} and the mutant TNF({Delta}1–12) genes were then subcloned into the CH269 vector and verified by sequencing.

E293 cells were transfected with the CH269 vector alone, wild-type TNF-{alpha}, mutant membrane-bound TNF-{alpha} or a combination of the LT-{alpha}(D50N)1ß2 and LT-ß constructs using Lipofectamine (Life Technologies) according to the manufacturer’s instructions. Cells were analyzed by flow cytometry for surface expression of LT-{alpha}(D50N)1ß2 and membrane-bound TNF-{alpha}. After 48 h, transfected E293 cells were cocultured with U1 monocytes at a 5:1 E:T cell ratio for 75 h. In addition, supernatants from the transfected E293 cells were used to culture U1 cells for 72 h, and then U1 supernatants were analyzed from triplicate wells for HIV-1 p24 Ag production by ELISA. To ensure that there was no secreted TNF-{alpha} in the supernatants of the E293 cells transfected with the mutant membrane-bound TNF-{alpha} plasmid, a TNF-{alpha} ELISA was performed on these supernatants according to the specifications of the manufacturer (Endogen, Woburn, MA).

The LT-ßRFc (2.5 µg/ml), anti-TNF-{alpha} cA2 Ab (2.5 µg/ml), and control human IgG1 (2.5 µg/ml) were preincubated with E293 cells. To exclude the presence of secreted or shed surface LT-{alpha}(D50N)1ß2 ligand that could stimulate p24 expression, supernatants from cells treated as described above were assayed for LT cytotoxic activity using a HT29 cytotoxicity assay as previously described (38).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LT-{alpha}1ß2 stimulates HIV-1 replication in the U1 monocytic cell line

To test whether LT-{alpha}1ß2 could induce HIV-1 replication, we stimulated U1 cells with soluble LT-{alpha}1ß2 and quantified p24 Ag levels as a measure of HIV-1 replication. As shown in Fig. 1Go, stimulation of U1 cells with LT-{alpha}1ß2 at a concentration of 10 ng/ml resulted in a 2.8-fold increase in HIV-1 replication. We note that concentrations of LT-{alpha}1ß2 between 10–50 ng/ml are in the linear range of the LT-{alpha}1ß2 dose response of U1 cells (data not shown). Furthermore, a kinetic analysis of LT-{alpha}1ß2 stimulation of HIV-1 replication demonstrated a sustained increase in p24 levels in LT-{alpha}1ß2-stimulated U1 cells on days 7, 14, 18, and 21 poststimulation (data not shown). By contrast, TNF-{alpha} treatment (1 ng/ml) of the cells resulted in peak p24 production on day 5, which was sustained for a total of 14 days (data not shown).



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  		FIGURE 1. LT-{alpha}1ß2 stimulates HIV-1 replication in the U1 monocytic cell line. The promyelomonocytic cell line, U1, that is chronically infected with HIV-1, was grown as previously described (34). Cells were treated with LT-{alpha}1ß2 (38) at 10 ng/ml or approximately 100 pM, the half-maximal stimulatory concentration of LT-{alpha}1ß2 in these experiments, and p24 Ag levels were measured 48 h later. The anti-TNF-{alpha} Ab cA2 at 5 µg/ml or the LT-ßR-blocking Ab (LT-ßRFc-CH2-less; 5 µg/ml) was added to the cells as indicated. There was no difference in cellular viability as determined by trypan blue exclusion between LT-{alpha}1ß2-treated and untreated U1 cells. The results shown are consistent with three independent experiments. The error bars represent the SD between duplicate values in the experiment displayed. By flow cytometric analysis (FACS) we demonstrated that the U1 promyelomonocytic cell line expresses the LT-ßR (data not shown). We note that addition of LT-{alpha}1ß2 at a concentration of 10 ng/ml resulted in an average 3- to 10-fold increase in HIV-1 replication dependent upon the particular U1 clone tested.

 
To verify that stimulation of HIV-1 by LT-{alpha}1ß2 was mediated specifically by the LT-{alpha}1ß2 ligand and not by LT-{alpha}3, a potential contaminant that binds to and signals through the two TNF receptors but not through the LT-ßR (46), we employed a LT-ßR decoy module. The LT-ßR decoy molecule is a LT-ßR Ig Fc fusion protein that binds to and blocks the function of LT-{alpha}1ß2, but not LT-{alpha}3 (46). As shown in Fig. 1Go, the LT-ßR decoy molecule blocked LT-{alpha}1ß2-mediated stimulation of HIV-1 replication, whereas a mAb to TNF-{alpha} did not. Thus, the LT-{alpha}1ß2 effect is not secondary to stimulation of the TNF receptors through an LT-{alpha}3 contaminant or by TNF-{alpha} itself (9, 33). These results establish an independent pathway for LT-{alpha}1ß2 signaling of HIV-1 in infected U1 cells.

LT-{alpha}1ß2 and TNF-{alpha} stimulate HIV-1 replication in a synergistic manner

The effect of adding soluble TNF-{alpha} together with LT-{alpha}1ß2 to U1 cells was up to 10-fold greater than the effect of adding either cytokine alone (Fig. 2Go). This functional cooperation can be blocked by an Ab to TNF-{alpha} (data not shown) or by TNFR55-Fc, a TNF-{alpha} receptor Fc decoy molecule (Fig. 2Go). Treatment with either of these TNF-{alpha} Abs resulted in a decrease in p24 production to the level expected with LT-{alpha}1ß2 stimulation alone. Similarly, the LT-ßR-Fc decoy molecule blocked the cooperative effect of LT-{alpha}1ß2 and TNF-{alpha} to the level of p24 production expected in U1 cells stimulated by TNF-{alpha} alone. Stimulation of HIV-1 replication by the combination of TNF-{alpha} and LT-{alpha}1ß2 could be completely abrogated by treating the cells with a combination of the LT-ßR-Fc and TNFR-Fc decoy molecules (Fig. 2Go). Thus, LT-{alpha}1ß2 and TNF-{alpha} act cooperatively to stimulate HIV-1 replication.



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  		FIGURE 2. LT-{alpha}1ß2 and TNF-{alpha} stimulate HIV-1 replication in a synergistic manner via distinct pathways. U1 cells were grown in medium alone or in medium treated with TNF-{alpha} (1 ng/ml) or LT-{alpha}1ß2 (10 ng/ml) plus TNF-{alpha} (1 ng/ml). Cellular supernatants were harvested 48 h after treatment, and p24 Ag levels were measured in duplicate samples. The blocking of HIV-1 replication stimulated by the combination of LT-{alpha}1ß2 (10 ng/ml) and TNF-{alpha} (1 ng/ml) was achieved with 5 µg/ml TNFR55-Fc (TNFR-Fc, which contains the 55-kDa TNF receptor fused to the Fc of human IgG1) or with 5 µg/ml LT-ßRFc. The results shown are consistent with three independent experiments. The error bars represent the SD between duplicate values in the experiment displayed. We note that concentrations of TNF-{alpha} >50 pM are cytotoxic to U1 cells by trypan blue exclusion assay and to other chronically infected cell lines (10). Therefore, 1 ng/ml (~10 pM) TNF-{alpha} was used, since it was not cytotoxic as assayed by trypan blue exclusion on day 2 (data not shown).

 
Surface LT-{alpha}1ß2 and TNF-{alpha} stimulate HIV-1 replication in U1 cells

Since the functional form of LT-{alpha}1ß2 is membrane anchored (14, 37, 47), and TNF-{alpha} occurs in both a membrane-bound and a soluble form, we next examined whether cellular contact with membrane-bound forms of LT-{alpha}1ß2 and TNF-{alpha} also increased HIV-1 replication (Fig. 3Go). We constructed expression vectors encoding LT-{alpha}1ß2, wild-type TNF-{alpha}, and a mutant TNF-{alpha} molecule bearing a disruption in the TNF-{alpha} proteolytic cleavage site rendering it membrane bound (12) and incapable of secretion to a soluble form. These vectors were transiently transfected into E293 cells, and surface expression of the molecules was confirmed by FACS analysis. Similar to previous results (45), at least 50% of the cells were transfected and, in general, >75% were transfected with and expressed the indicated construct (data not shown).



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  		FIGURE 3. Soluble and membrane-bound forms of LT-{alpha}1ß2 and TNF-{alpha} stimulate HIV-1 replication identically. U1 cells were cocultured with E293 cells transfected with wt-TNF, mTNF, or membrane-bound LT{alpha}(D50N)1ß2 (mLT{alpha}ß) expression vector, and p24 levels were measured in the supernatants after 48 h of coculture. All data are presented as the mean ± SEM of at least three independent experiments. A, Membrane-bound LT-{alpha}1ß2 stimulates HIV replication. U1 cells were cocultured with E293 cells transfected with the membrane-bound LT{alpha}(D50N)1ß2 (mLT{alpha}ß) expression vector and treated with either 5 µg/ml LTßR-Fc or 5 µg/ml of an irrelevant Ig-matched control Ab. B, Membrane-bound and soluble TNF-{alpha} identically stimulate HIV-1 replication. U1 cells were cocultured with E293 cells transfected with wt-TNF, mTNF, membrane-bound LT{alpha}(D50N)1ß2 (mLT{alpha}ß) expression vectors, or combinations thereof as noted. The anti-TNF-{alpha} Ab cA2 (5 µg/ml) was used to block the effect of TNF-{alpha} on HIV-1 replication. E293 cells transfected with the vector encoding the wt-TNF-{alpha} molecule expressed both membrane-bound (as determined by FACS; data not shown) and soluble TNF-{alpha} as shown in C and D. C and D, Membrane-bound LT-{alpha}1ß2 and TNF-{alpha} are not secreted. C, Supernatants of transfected E293 cells were incubated with U1 cells, and p24 levels were measured after 48 h. D, Membrane-bound LT-{alpha}1ß2 and TNF-{alpha} are not secreted. Forty-eight hours after transfection, TNF-{alpha} levels were measured in the supernatants of transfected E293 cells by ELISA.

 
To ensure that there was no contamination by LT-{alpha}3 in the coculture experiment with the LT-{alpha}1ß2-expressing E293 cells, we cotransfected the LT-ß expression vector along with the expression vector bearing the previously characterized LT-{alpha} mutation D50N, LT-{alpha}(D50N). The (LT-{alpha}(D50N)) mutation abolishes the ability of LT-{alpha} homotrimers to bind to the TNF-p55 and TNF-p75 receptors, and hence, any potential LT-{alpha}3 breakdown product or LT-{alpha}1ß2 in E293 supernatants cannot signal (38). We note that the ability of the LT-{alpha}(D50N)1ß2 molecule to bind to and signal through the LT-ßR (38) is not affected. Cocultivation of E293 cells expressing LT-{alpha}(D50N)1ß2 (designated LT-{alpha}ß in the figure) stimulated HIV-1 replication in contiguous U1 monocytes, and the LT-ßR decoy molecule specifically blocked induction of p24 production (Fig. 3GoA). Identical results were obtained in a cocultivation experiment using insect cells expressing LT-{alpha}(D50N)1ß2 following infection with a baculovirus vector encoding LT-{alpha}(D50N)1ß2 (data not shown). These experiments demonstrate that signaling through the LT-ßR via the membrane-bound LT-{alpha}1ß2 ligand stimulates HIV-1 replication.

Cocultivation of E293 cells expressing either the wild-type and, therefore, secretable TNF-{alpha} (wt-TNF) or the membrane-bound TNF-{alpha} (mTNF) molecule stimulated HIV-1 replication in U1 cells to a similar degree (Fig. 3GoB). In both cases the specificity of this effect was demonstrated by the use of a TNF-{alpha} mAb that abrogated p24 induction, whereas a control IgG1 isotype-matched Ab had no effect (Fig. 3GoB).

When E293 cells expressing the wt-TNF-{alpha} or the membrane-bound TNF-{alpha} form were combined with E293 cells expressing membrane-bound LT-{alpha}1ß2 and cocultivated with U1 cells, we observed a similar enhancement of p24 production with both the soluble and membrane-bound molecules (Fig. 3GoB). We did not detect an increase in the apoptosis of U1 cells cocultivated with the cells expressing membrane-bound TNF-{alpha} versus those expressing wt-TNF-{alpha} using flow cytometric analysis of subdiploid DNA as an apoptotic marker (48) or in cellular viability as measured by trypan blue exclusion (data not shown).

To verify that there was no secreted TNF-{alpha} or LT-{alpha} in the supernatants of the E293 cells that expressed the membrane-bound LT or TNF-{alpha} forms, we cocultivated U1 cells with supernatants from E293 cells expressing these molecules and measured p24 levels. As expected, exposure of U1 cells to cellular supernatants from E293 cells expressing the secretable wt-TNF-{alpha} resulted in the stimulation of HIV-1 replication (Fig. 3GoC). Supernatants from cells transfected with either the membrane-bound TNF-{alpha} or LT molecules did not cause an increase in p24 levels (Fig. 3GoC). Consistent with these findings, an ELISA of the supernatants measuring TNF-{alpha} levels demonstrated that soluble TNF-{alpha} was present in the supernatants from E293 cells transfected with the wt-TNF-{alpha} construct but not in the supernatants of cells transfected with the membrane-bound TNF-{alpha} or LT-{alpha}ß constructs (Fig. 3GoD). Thus, specific engagement of the TNF-{alpha} and LT-ß receptors by their respective membrane-bound ligands caused the observed up-regulation of p24 production.

Clustering of LT-ßR and TNFp75R cooperatively enhances HIV-1 replication

TNF-{alpha} appears to signal through the clustering of its cognate receptors, p55 and p75 (11). Similarly, an anti-LT-ßR agonist Ab, which clusters the LT-ßR, can elicit LT-ßR-mediated biological functions in tissue culture (39, 48). As shown in Fig. 4Go, specific clustering of the LT-ßR on the surface of U1 cells also stimulated HIV-1 replication, as did clustering of the p75 TNF receptor. By contrast, a control Ig-matched Ab did not increase p24 levels above baseline (data not shown). Notably, simultaneous clustering of both LT-ßR and p75 TNF receptor resulted in a marked enhancement of HIV-1 replication compared with that after engagement of either receptor alone. Thus, soluble and membrane-bound forms of LT-{alpha}1ß2 and TNF-{alpha} or the specific engagement of their cognate receptors result in cooperative stimulation of HIV-1 replication.



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  		FIGURE 4. Clustering of LTß-R and p75 TNF-{alpha} receptor synergistically stimulates HIV-1 replication. Affinity-purified goat anti-IgG Fc was immobilized on 24-well plates, incubated with the Abs indicated, and overlaid with U1 cells for 48 h, and the HIV-1 p24 Ag concentration was measured. As a control for U1 cell responsiveness, stimulation of p24 production by soluble LT-{alpha}1ß2 ligand at 50 ng/ml was also performed. The results shown are consistent with three independent experiments. The error bars represent the SD between duplicate values in the experiment displayed. We note that U1 cells undergo a time-dependent decay in the ability of LT-{alpha}1ß2 to induce HIV-1 replication as noted previously for TNF-{alpha} (34). Thus, addition of LT-{alpha}1ß2 at a concentration of 50 ng/ml resulted in variable activation of HIV-1 replication in the U1 clone similar to that after addition of the ligand at 10 ng/ml (see Fig. 1Go).

 
TNF-{alpha}, but not LT-{alpha}1ß2, induces NF-{kappa}B binding activity in U1 cells

TNF-{alpha} causes the nuclear translocation of NF-{kappa}B (reviewed in 49), and the binding of NF-{kappa}B to the HIV-LTR is thought to mediate TNF-{alpha}-induced HIV-1 gene transcription and replication in U1 cells (33). In some cell lines LT-{alpha}1ß2 also causes the nuclear translocation of NF-{kappa}B (48, 50). Thus, we explored whether the mechanism of LT-{alpha}1ß2-mediated stimulation of HIV-1 replication in U1 cells involved NF-{kappa}B. Specifically, we stimulated U1 cells with LT-{alpha}1ß2 and/or TNF-{alpha}, prepared nuclear extracts, and performed an EMSA using an NF-{kappa}B site from the HIV-1 LTR as a probe. As shown in Fig. 5Go, TNF-{alpha} caused the induction of NF-{kappa}B binding activity (lanes 2 and 4), whereas treatment with LT-{alpha}1ß2 at concentrations that stimulate an increase in p24 production did not result in detectable inducible NF-{kappa}B binding activity (lane 3). Thus, the observed cooperation between LT-{alpha}1ß2 and TNF-{alpha} raises the possibility that another undefined HIV-1 transcriptional activation pathway is stimulated via the LT-ßR receptor.



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  		FIGURE 5. TNF-{alpha}, but not LT-{alpha}1ß2, stimulates NF-{kappa}B binding activity in U1 cells. An EMSA using nuclear extracts from unstimulated UI cells (un) or cells stimulated with 100 U/ml TNF-{alpha} and/or 50 ng/ml LT-{alpha}1ß2 for 2 h as indicated. A TNF-{alpha}-inducible complex binds to the HIV-1-NF{kappa}B site oligonucleotide probe (lanes 1 and2), whereas there is no inducible complex observed after LT-{alpha}1ß2 stimulation. An oligonucleotide probe matching the sequences spanning between -61 and -28 relative to the human TNF-{alpha} cap site containing an Sp1 site was used as a control for protein loading and processing of the EMSA. The experiment displayed is representative of three experiments performed in three independent U1 clones. We note that HIV-1 p24 levels were inducible by both TNF-{alpha} and LT-{alpha}1ß2 in all three clones (data not shown).

 
HIV-1 infection stimulates LT-{alpha}1ß2 expression on the surface of H9 T cells.

To determine whether HIV-1 infection up-regulates LT-{alpha}1ß2 expression on T cells, we screened a panel of T cell lines for the effect of HIV-1 infection on surface expression of LT-{alpha}1ß2. We infected the T cell hybridoma II-23 and the T cell lines CEM, Jurkat, and H9 with the HIV-1 MN strain at a multiplicity of infection of 1.0 and analyzed these cells 4 days later by flow cytometry for surface LT-{alpha}1ß2 expression. HIV-1 infection did not induce LT-{alpha}1ß2 surface expression on CEM, Jurkat, or II-23 cells (data not shown). However, in the case of the H9 cell line, HIV-1 infection did cause a reproducible increase in LT-{alpha}1ß2 surface expression 4 days after infection (Fig. 6Go), whereas HIV-1 infection had no effect on surface TNF-{alpha} expression (data not shown). To demonstrate that the functional LT-{alpha}1ß2 heterotrimer is specifically up-regulated by HIV-1 infection, we stained H9 cells with the Abs NC2 and CH12. The NC2 Ab recognizes all LT-{alpha}-containing forms, whereas CH12 specifically recognizes the surface LT-{alpha}2ß1 forms. As shown in Fig. 6Go, the NC2 Ab, but not the CH12 Ab, recognizes the inducible LT complex, indicating that LT-{alpha}1ß2 is up-regulated by HIV-1 infection of H9 cells.



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  		FIGURE 6. HIV-1 infection stimulates LT-{alpha}1ß2 expression on the surface of T cells. Flow cytometric analysis of HIV-1 infected T cells (HIV+) was performed on day 4 postinfection. HIV(-) curves denote LT-{alpha}1ß2 expression on uninfected H9 cells. The left-most curve represents negative control staining with isotype-matched control and secondary FITC-labeled Ab. In the top panel, cells were stained with LT-ßRFc followed by FITC-labeled goat anti-human Ig. In the middle and bottom panels, the Abs NC2 and CH12, which respectively recognize either all LT-{alpha}-containing forms or only the LT-{alpha}2ß1 form of surface LT, were used. The experiment displayed is representative of eight independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that HIV-1 replication can be triggered by LT-ßR activation alone and that it is cooperatively stimulated when the distinct LT-ßR and TNF receptor systems are simultaneously engaged on monocytes. Monocytes and the monocytic dendritic/reticular network of cells bear the CCR5 chemokine receptor, which is used by HIV-1 for cellular entry (51, 52, 53). In the secondary lymphoid tissues, dendritic cells can sequester up to 90% of the total HIV-1 viral burden on their cellular surfaces (30) and are important in transmitting HIV-1 to T cells (54). Thus, our demonstration that LT-{alpha}1ß2 and TNF-{alpha} cooperatively stimulate HIV-1 replication may suggest an important host mechanism for regulating HIV-1 viral burden in monocytic cells, which are critical to HIV-1 infection and progression.

Interestingly, activation of the LT-ßR potentiates TNF-{alpha}-mediated cell death (55), providing a precedent for cooperative interactions between the two receptor systems. In certain cell types, LT-ßR signaling results in NF-{kappa}B activation via the recruitment of TNF receptor-associated factor-3 (TRAF3) and -5 (56). NF-{kappa}B translocation secondary to TNFR p55 signaling involves TRAF2 (57), and TRAF1 is involved in TNFR p75-induced NF-{kappa}B modulation (58). We were not able to detect LT-ßR-mediated induction of NF-{kappa}B in U1 cells, suggesting that either the levels of NF-{kappa}B induced by the LT-ßR in U1 cells are too low for detection by EMSA or that HIV-1 stimulation by LT-ßR may be NF-{kappa}B independent. These results are consistent with the observation that synergistic activation of HIV replication by other cytokine combinations acts at different transcriptional and post-transcriptional steps in the HIV-1 life cycle (7). Notably, the LT-ßR-stimulated pathway differs from that mediated by the TNF receptors in that it is mediated by different intracellular TRAFs. Perhaps in the case of HIV-1 replication, the recruitment of a different set of TRAFs by the LT-ßR results in the activation of a distinct signal transduction pathway that is complementary to the signal transduction pathways activated by TNF-{alpha}.

The results of our cocultivation experiments with E293 cells expressing membrane-bound TNF-{alpha} are in contrast to results obtained in another study using CHO or 3T3 cells stably transfected with a membrane-bound form of TNF-{alpha} and then cocultured with latently infected ACH-2 T cells or HIV-1-infected PBLs (59). In this study the target cells appeared to preferentially undergo apoptosis and down-regulation of HIV-1 replication when they were cocultivated with a membrane-anchored form of TNF-{alpha}. The differences between this study and ours might be attributed to the fact that in the study by Lazdins et al. 100% of the stably transfected CHO and 3T3 cells would be expected to express membrane-bound TNF-{alpha}. Since a wt-TNF-{alpha}-expressing cell line was not employed in their cocultivation experiments as a control, it is not possible to determine whether the level of TNF-{alpha} expression in the transfected cells was toxic to the HIV-1-infected cells, thus causing cell death.

Since HIV-1-infected monocytes are particularly numerous in lymph nodes and spleen, where they are in intimate contact with T cells in the early stages of HIV-1 infection (31), it is intriguing to speculate that activated lymphocytes expressing surface LT-{alpha}1ß2 or TNF-{alpha} forms may play an important role in promoting HIV-1 replication in contiguous monocytes. Early HIV-1 infection is characterized by lymphadenopathy (29, 60), splenomegaly, and lymphocyte activation (61, 62, 63). In fact, disturbances of lymphoid organs are among the earliest changes observed in HIV-1 infection (31, 30, 64). However, as HIV-1 infection progresses with concomitant T cell death, the total lymphoid tissue in the infected host dramatically decreases (29, 65).

Interestingly, perturbations of the LT-{alpha}1ß2 and TNF-{alpha} ligand receptor systems also cause disturbances in the lymphoid cells and organs. The LT-ß signaling pathway is required for lymph node organogenesis and lymphoid architecture in the developing mouse, and the loss of LT-ß signaling in normal adult mice leads to an altered splenic marginal zone and the collapse of follicular dendritic cell (FDC) networks in both spleen and lymph nodes (18, 22). TNF-{alpha} is also involved in the generation of FDCs and the formation of splenic germinal centers and functions as an autocrine B cell growth factor (24, 25, 26, 27, 28).

Perhaps early in HIV-1 infection, the up-regulation of LT-{alpha}1ß2 together with membrane-bound TNF-{alpha} in HIV-1-infected lymphocytes acts to stimulate lymphoid proliferation and dendritic cell maturation. It is interesting to speculate that as rounds of HIV-1 replication are cooperatively driven by the TNF-{alpha} and LT-{alpha}1ß2 signaling pathways, and HIV-1 infection progresses to AIDS, the virus destroys LT-{alpha}1ß2-bearing T cells and a crucial lymphoid architectural signal is lost.


   Acknowledgments
 
We thank Katherine Hanify for technical assistance and those at Biogen whose efforts led to the development and characterization of the LT reagents. We thank Fabienne Mackay, Paula Hochman, and Evelyn Kurt-Jones for helpful discussions and Carl Ware, Bruce Walker, and anonymous reviewers for helpful comments on the manuscript. We also thank Centocor, Hoffmann-La Roche, Werner Lesslauer, George Kollias, and Jeff Bergelson for the generous gifts of reagents.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants CA58735 and P30CA06516-33 (to A.E.G.), AI28691-06 (to R.W.F.), and AI01078-03 (to W.L.M.); an Established Investigator Award from the American Heart Association Foundation (to A.E.G.); and a grant from the Stichting Dr. Catharina Van Tussenbroek (to B.M.N.B.). Back

2 Address correspondence and reprint requests to Dr. Anne E. Goldfeld, Center for Blood Research, 800 Huntington Ave., Boston, MA 02115. E-mail address: Back

3 Abbreviations used in this paper: LT-{alpha}1ß2, lymphotoxin-{alpha}1ß2; LT-ßR, lymphotoxin-ß receptor; LT-{alpha}3, lymphotoxin-{alpha} homotrimer; wt-TNF, wild-type TNF; mTNF, membrane-bound TNF; TRAF, TNF receptor-associated factor. Back

Received for publication June 26, 1998. Accepted for publication February 26, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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